A recent government study on the accuracy of field tests for anthrax spores has cast serious doubt on the validity of these tests. This report clearly indicates the urgent need for a more rigorous selection of deoxyribonucleic (DNA) markers for anthrax testing. The proposed project aims to develop a more specific nucleic acid test for anthrax spores. PCR primers will be designed to amplify sequences in the anthrax bacterial chromosome and plasmids that can differentiate anthrax from related species. Rapid extraction of nucleic acids from anthrax spores is also a challenge for field tests. We will develop efficient spore disruption and nucleic acid extraction techniques by exploring a combination of approaches, including germination induction and mechanical disruption. The nucleic acid extraction and amplification protocols will then be incorporated into our proprietary lab-in-a-tube (Liat (TM)) platform to develop a handheld device that is suitable for field tests. The Liat system has been shown to be capable of performing rapid close-tube polymerase chain reaction (ct-PCR) DNA amplifications in 1/6 the time required by conventional thermal cyclers. Moreover, the system can be operated for 6 hours on battery power. Novel approaches to combine multiple tests in a single tube that will surpass the capability of other diagnostic technology platforms are also under development. A closed-tube assay will be safer to implement and the hand-held device will deliver results more quickly than most of the systems currently in operation. This fully automated "sample-to-answer" system will enable minimally trained operators to perform sophisticated nucleic acid tests in the field.